Browsing by Subject "Rho"

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Open Access
Identification of a Novel Formin-GAP Complex and Its Role in Macrophage Migration and Phagocytosis
(2011) Mason, Frank Marshall
Essential and diverse biological processes such as cell division, morphogenesis and migration are regulated by a family of molecular switches called Rho GTPases. These proteins cycle between active, GTP-bound states and inactive, GDP-bound state and this cycle is regulated by families of proteins called Rho GEFs and GAPs. GAPs are proteins that stimulate the intrinsic GTPase activity of Rho-family proteins, potentiating the active to inactive transition. GAPs target specific spatiotemporal pools of GTPases by responding to cellular cues and utilizing protein-protein interactions. By dissecting these interactions and pathways, we can infer and then decipher the biological functions of these GAPs.
This work focuses on the characterization of a novel Rho-family GAP called srGAP2. In this study, we identify that srGAP2 is a Rac-specific GAP that binds a Formin-family member, Formin-like 1 (FMNL1). FMNL1 is activated by Rac and polymerizes, bundles and severs actin filaments. srGAP2 specifically inhibits the actin severing of active FMNL1, and the assembly of an srGAP2-FMNL1 complex is regulated by Rac. Work on FMNL1 shows that it plays important roles in regulating phagocytosis and adhesion in macrophages. To learn more about srGAP2 and its role in regulating FMNL1, we studied macrophages isolated from an srGAP2 KO mouse we have recently generated. This has proven quite fruitful: loss of srGAP2 decreases the ability for macrophages to invade through extracellular matrix but increases phagocytosis. These results suggest that these two processes might be coordinated in vivo by srGAP2 and that srGAP2 might be a critical regulator of the innate immune system.
Open Access
Using Light to Control Protein-Protein Interactions: Optogenetics in Drosophila melanogaster
(2016-04-23) Lo, Alexis
Recent advancements in genetically encoded light-sensitive protein systems, also known as optogenetic systems, have stemmed from the many benefits of using blue light stimuli to selectively initiate protein-protein interactions. Such benefits include the non-invasive nature of light, the precision of the stimulus, and the reversibility of the protein-protein interactions in the dark. One specific optogenetic system from Arabidopsis thaliana, the CRY2/CIB module, offers a powerful genetically encoded mechanism by which to study the role of proteins in a tissue-specific manner during various stages of development. Using cloning techniques to generate CRY2 and CIB constructs in Drosophila specific vectors, we have attempted to adapt the CRY2/CIB system to Drosophila. We tested an oligomerizing version of the CRY2 component as a tool for the negative regulation of targeted proteins in Drosophila. Although we were unable to repeat the clustering results observed in yeast, we worked on modifying our light activation protocol and discovered the sensitivity of the system to inadvertent light stimulation during preparation for imaging. We also conducted cloning in order to perform a proof-of concept experiment utilizing both cytoplasmically diffuse CRY2 and membrane-anchored CIBN. Thus far, germline transformants of the CIBN component have been generated, and work will continue to generate the CRY2 germline transformants. Additionally, we are also working on cloning variants of the small G protein Rho to form a fusion protein with the CRY2 component. At the plasma membrane, Rho proteins catalyze signaling cascades to affect actin and myosin formation and cytoskeletal changes. If Drosophila Rho1 proteins are successfully adapted to CRY2 components, upon blue light stimulation the recruitment of CRY2 to a CIB component anchored in the membrane could be spatially and temporally controlled to affect subsequent downstream events. The ability to drive Rho1 to the membrane at specific stages of development will generate a better understanding of the effects of altering cytoskeletal function during Drosophila morphogenesis and thereby give insight into wound healing and tissue regeneration processes in vertebrates.